The long term objectives are to elucidate the molecular mechanisms which regulate the amounts and types of membrane phospholipids synthesized to meet the demands for membrane assembly and maintenance, for lipoprotein and bile formation, for lung surfactant synthesis, milk fat globule formation, for energy storage, and for formation of bioregulators (e.g. sn1,2diacylglycerols). The experimental approach is one of biochemistry and molecular genetics. Each specific aim focuses on a key enzyme involved in membrane phospholipid biosynthesis and its structural and regulatory genes. During the last grant period, the E. coli p1sB gene was sequenced, its snglycerol3P acyltransferase gene product was purified, chemically characterized, and reconstituted. Two dimensional crystals were characterized. The specific aims on the glycerol-P acyltransferase are to characterize the homogeneous enzyme kinetically in mixed micelles, and in vesicles, to elucidate specificity and stoichiometry of the phospholipid cofactor requirement and mechanism of action; to further the structural analysis of the 2D crystalline tubular arrays by electron microscopic techniques; and to further knowledge of function and regulation by genetic techniques. Specific Aim 2 focuses on the isolation, sequence analysis and expression of the glycerolP/DHAP acyltransferase sturctural gene of Saccharomyces cerevisiae. These studies will utilize mutants which were isolated during the last grant period. Studies on solubilization, purification, characterization and regulation at the level of gene expression and enzyme activity are proposed. The third, and final specific aim, focuses on the isolation, sequence analysis, expression and regulation of the diacylglycerol choline- and ethanol amine-phosphotransferase structural genes and gene products of Saccharomyces cerevisiae. Mutants have been selected and complemented with hybrid plasmids. The genetic analysis will allow regulation at the sn-1,2-diacylglycerol branch point to be investigated. The structures of the gene and gene products of these critical enzymes will be determined. The regulation of phospholipid and macromolecular syntheses will be investigated using each type of mutant. The studies are of fundamental importance to significant health problems (cancer, cardiovascular and digestive diseases; obesity, hypertension and stroke; respiratory distress syndrome and alcholism).